Bacillus thuringiensis, a spore-forming bacterium of which there are more than 200 naturally occurring variants, produces a rhombic crystal during sporulation. This crystal is toxic upon ingestion to a wide variety of lepidopteran larvae. Many of these susceptible larvae are economically important crop pests. The toxic factor in the crystal is derived from a protein protoxin of molecular weight 130,000 which has been termed the .delta.-endotoxin; the protoxin is not in itself toxic but requires proteolytic processing to yield an active toxin (activated .delta.-endotoxin),and processing normally occurs in the insect gut.
Bacillus thuringiensis (B.t.) toxin has provided a basis for commercial formulations of insecticide for at least ten years. The active ingredient in these products is dried preparations of sporulated B.t. cells. Included in this dried powder is the rhombic crystal and the viable spore which can regenerate to give rise to vegetative B.t. cells.
The commercial formulations of B.t. have found use in markets ranging from vegetables to forest management. Limitations to the use of this product, however, include:
1. Instability of the toxin gene in the micro-organism results in difficulties in quality control. PA1 2. Problems in application arise, as the toxin is in particulate rather than soluble form. PA1 3. Speed of action is slower than that of chemical pesticides, possibly because of the requisite protoxin to toxin activation step. PA1 4. Manifestation of toxicity requires ingestion of the toxic crystal by a susceptible insect. PA1 5. Commercial preparations contain viable spores. PA1 1. The toxin is highly specific to target insect pests and is inactive against other life forms, including beneficial insects. PA1 2. The active factor is a protein molecule and, as a consequence, is rapidly biodegradable and poses little risk of long term impact on the ecosystem. PA1 1. The gene is more stable than its counterpart in B.t. and may be more stable than the relatively undefined fragment of DNA cloned by either Schnepf et al (Schnepf, H. E. and Whiteley, H. R., U.S Pat. No 4,448,885) or Klier et al. (Klier, A., Rapoport, G., Dedonder, R [Filing date Apr. 26, 1982] Demande de Brevet Europeen 0 093 062). The instability of the naturally occurring gene may arise because it lies within a transposon-like structure (Lereclus, D., Ribier, J., Klier, A., Menou, G. and Lecadet, M. M. [1984] EMBO Journal 3:2561-2567). Inverted repeat sequences that are characteristic of a transposon have been eliminated from the plasmid of the subject invention. PA1 2. The activated .delta.-endotoxin produced directly from the hybrid gene is, in essence, a chemical product. The formulation in which it is applied for pest control will contain no viable microorganisms or spores. This constitutes a significant advantage over the commercial preparations presently in use that result in the application of viable spores into the environment. PA1 3. The activated .delta.-endotoxin produced by the hybrid gene is insoluble but is readily extracted into soluble form in aqueous solutions. This can present advantages for application. Insoluble toxin crystals derived from B.t. may present problems with regard to application and coverage. These problems are obviated with a soluble preparation. PA1 4. The theoretical rate of expression is increased at least two-fold over the constructions patented by either Schnepf, et al. (Schnepf, H. E. and Whiteley, H. R., U.S. Pat. No. 4,448,885) or Klier et al. (Klier, A., Rapoport, G., Dedonder, R. [Filing date Apr. 26, 1982] Demande de Brevet Europeen 0 093 062), as the recombinant gene product of the hybrid gene is only half as large as the entire protoxin. The advantages may be greater than this as the C-terminal half of the molecule, which has been eliminated in the construction, may result in a growth/expression limitation in E. coli. PA1 5. The specific activity of the activated molecule produced by the hybrid gene is effectively double that of the protoxin, as the half of the molecule that makes no contribution to toxicity has been eliminated. Thus, on a weight basis, the activity of the material is about twice that of the protoxin produced by the recombinant plasmid of either Schnepf et al. (Schnepf, H. E. and Whiteley, H. R., U.S. Pat. No. 4,448,885) or Klier et al. (Klier, A., Rapoport, G., Dedonder, R. [Filing date Apr. 26, 1982] Demande de Brevet Europeen 0 093 062). PA1 6. The protein produced by the hybrid gene is a preactivated toxin and requires no further processing or alteration for full activity. In contrast, the .delta.-endotoxins derived from the natural source as well as those expressed by the recombinant plasmid of others (Schnepf, H. E. and Whiteley, H. R., U.S. Pat. No. 4,448,885):Klier, A., Rapoport, G., Dedonder, R. [Filing date Apr. 26, 1982] Demande de Brevet Europeen 0 093 062) are inactive molecules and require proteolytic processing for activity (Lecadet, M. M. and Dedonder, R. [1967] J. Invert. Pathol. 9:322). Although processing of these protoxins can occur in the insect gut, this preactivation may provide an improvement in speed of kill, an important consideration in the commercial utilization of B.t. toxin.
In spite of these limitations, use of B.t. preparations has been increasing, particularly in such areas as forest pest management The reasons for this include:
In 1981, a gene encoding the protoxin from a commercial strain of B.t. was cloned and expressed in E. coli by Schnepf and coworkers (Schnepf, H. E. and Whiteley, H. R. [1981] Proc. Natl. Acad. Sci USA 78: 2893-2897) A U.S. patent was granted on this construction (U.S. Pat. No. 4,448,885). The recombinant plasmid encodes the entire protoxin molecule and the gene is under the control of its natural promoter. Subsequently, a European patent has been filed by Klier et al on a recombinant protoxin gene from what is presumably a different strain (B. thuringiensis 1715) (Klier, A., Rapoport, G., Dedonder, R. [Filing date Apr. 26, 1982] Demande de Brevet Europeen 0 093 062). In neither of these patents is the sequence of the gene or the protein product disclosed It is clear that the toxin genes in both cases are bounded by undefined sequences of DNA.